Pathogenesis and pathophysiology of inherited carnitine deficiency using carnitine-deficient JVS mice

肉碱是线粒体脂肪酸氧化的必需成分幼年内脏脂肪变性(JVS)小鼠患有脂肪肝、高氨血症和低血糖，1988年在金泽大学被发现是肉碱缺乏的。我们描述了高氨血症是由于尿素循环酶的减少而导致的，而尿素循环酶是由于基因转录被抑制而导致的。此外，我们还发现JVS小鼠也表现出心肌肥大。在本研究中，我们对JVS小鼠的发病机制和病理生理学进行了研究。JVS小鼠的主要缺陷是肉碱在肾脏的转运，这是钠依赖的，对肉碱有很高的亲和力，并被肉碱类似物D-肉碱和γ-丁甜菜碱抑制。JVS小鼠尿液中有大量的γ-丁基甜菜碱排泄。由于伽马-丁甜菜碱是肉碱生物合成的直接前体，而且由于最后一步酶，伽马-丁甜菜碱…JVS小鼠体内的羟基酶活性较高，说明肉碱的生物合成途径是正常的，但由于JVS小鼠对前体的重吸收有缺陷，肉碱的生物合成途径存在继发性缺陷。通过与Kanazawa小组的合作研究，我们将JVS基因定位在11号染色体上D11Mit31微卫星座位附近。JVS小鼠尿素循环酶基因的表达受到抑制，并伴随着其他几个肝脏特异性酶或蛋白基因的表达抑制，如酪氨酸氨基转移酶和白蛋白。这些都是由糖皮质激素引起的。JVS组小鼠血清糖皮质激素水平明显高于THA对照组，其核团中含有丰富的糖皮质激素受体蛋白。另一方面，通过凝胶移位实验检测到核转录因子AP-1在JVS小鼠中非常活跃，并在断奶期间成为活性因子。根据这些结果，我们推测AP-1与糖皮质激素受体相互作用，从而抑制了糖皮质激素对许多基因的诱导。为了将JVS小鼠肝脏中肉碱缺乏与AP-1激活联系起来，我们检测了脂肪酸和脂肪酸肉碱对原代培养的肝细胞糖皮质激素诱导的氨基甲酰磷酸合成酶的影响。油酸、花生四烯酸等长链不饱和脂肪酸可抑制糖皮质激素诱导的CPS。加入肉碱后，脂肪酸的抑制作用几乎完全消失，提示原代培养的肝细胞由于肉碱缺乏而不能代谢长链脂肪酸，与肉碱缺乏的JVS小鼠相似。似乎是不饱和长链脂肪酸引起AP-1的激活。我们发现心肌肥厚的部分原因是由儿茶酚胺代谢的激活所介导的。几种阻滞剂抑制儿茶酚胺代谢可部分抑制心肌肥厚。为了更准确地阐明心肌肥厚的病理生理机制，我们采用差异显示技术对正常和肥厚的心室中表达的mRNAs进行了差异显示，发现了几种未知的mRNAs在不同的脑室中表达存在差异。较少

Carnitine is an essential component for the oxidation of fatty acid in mitochondria Juvenile visceral steatosis (JVS) mice suffering from fatty liver, hyperammonemia and hypoglycaemia, discovered at Kanazawa University in 1988, were found to be carnitine-deficient. We described that the hyperammonemia is caused by the decrease of the urea cycle enzymes which results from suppressed transcription of the genes. Furthermore, we found that JVS mice show cardiac hypertrophy, too. In this study, we focused our researches on the pathogenesis and pathophysiology of JVS mice. The primary defect of JVS mice was in the transport of carnitine in the kidney, which was sodium-dependent with a high affinity for carnitine and was inhibited by carnitine analogues, D-carnitine and gamma-butyrobetaine. Large amounts of gamma-butyrobetaine were excreted in the urine of JVS mice. Since gamma-butyrobetaine is the direct precursor of carnitine biosynthesis and since the last step enzyme, gamma-butyrobetaine … More hydroxylase, was rather higher in JVS mice, we conclude that the biosynthesis pathway of carnitine is normal but secondarily defective due to defective reabsorption of the precursor in JVS mice. By the cooperative study with Kanazawa group, we mapped the jvs gene on chromosome 11, in the vicinity of microsatellite locus D11Mit31.The suppressed expression of the urea cycle enzyme genes in JVS mice was accompanied with that of several other liver-specific enzyme or protein genes such as tyrosine aminotransferase and albumin. Those are all induced by glucocorticoid. Serum glucocorticoid levels of JVS mice were rather higher than tha controls and glucocorticoid receptor protein was abundant in the nuclear fraction. On the other hand, a nuclear transcription factor, AP-1, detected by gel-shift assay, was found to be very active in JVS mice and became activer during the weaning period. From these results, we reason that AP-1 interacts with glucocorticoid receptor and as the result, suppresses the induction by glucocorticoid of many genes. To connect the carnitine deficiency and AP-1 activation in the liver of JVS mice, we tested the effect of fatty acid and fatty acid+carnitine on the glucocorticoid induction of carbamylphosphate synthetase in the primary cultured hepatocytes. Long-chain unsaturated fatty acid, such as oleic acid and arachdonic acid, suppressed the induction of CPS by glucocorticoid. The suppression by fatty acids almost completely disappeared by the addition of carnitine, which suggests that the primary cultured hepatocytes can not metabolize long-chain fatty acid due to carnitine deficiency and mimic the situation of carnitine-deficient JVS mice. It seems that unsaturated long-chain fatty acids causes activation of AP-1. We found that some part of the cause of the cardiac hypertrophy was mediated by activation of catecholamine metabolism. The suppression of catecholamine metabolism by several inhibitors caused a partial suppression of cardiac hypertrophy. To clarify more precisely the pathophysiology of the cardiac hypertrophy, we adopted differential display for mRNAs expressed in normal and hypertrophied ventricles and found several unknown mRNA species expressed differently between the ventricles. Less